Some of the cytosine residues in all vertebrate genomes are methylated, producing what amounts to a fifth DNA base, 5-methylcytosine. Methylation has been shown to regulate gene expression, and hence alter cellular state. Differentiation and development of genetically identical cells seems to be controlled by a combination of signaling and epigenetic effects, including DNA methylation state. In addition, several types of disease have shown a direct dependence on methylated state, e.g. colorectal or breast cancer. The relevance of DNA methylation in disease state, cellular control, growth and differentiation illustrates the importance of techniques capable of measuring or detecting DNA methylation.
In particular, techniques are needed to detect methylated DNA within mixtures of DNA including DNA having different methylation levels or no methylation. For example, sorting and isolating methylated DNA facilitates further analysis of DNA sequence, methylation pattern or content, including determining which regions are methylated (e.g., islands of hypermethylation) and/or unmethylated (e.g., regions of hypomethylation). Thus, techniques that can reliably, efficiently and sensitively detect, measure, classify, or separate DNA based on DNA methylation have application in a number of fields, including but not limited to health, medicine, animal and plant husbandry. Methods provided herein have use in other fields, including for example, targeting drugs, screens or assays for diseases, disease states and predisposition toward a disease.
Conventional processes known in the art separate methylated from unmethylated DNA either through immunoprecipitation of methylated DNA (MeDIP), methylation specific binding protein columns, or methylation-sensitive restriction digestion. Immunoprecipitation suffers from a lack of specificity, in that unmethylated fragments are often co-precipitated with the methylated fragments, causing those techniques to only be sensitive to large changes in methylation, restricting the technique to examining highly methylated regions. A similar problem occurs with methylation binding protein columns, as unmethylated fragments are often bound as well, and the strength of binding may vary depending on the degree of methylation. Dense methylation is required for strong, specific binding, causing the same type of problem as with MeDIP. Alternatively, methyl-sensitive restriction digestion requires relatively long intact DNA fragments, and is limited to CGs inside the recognition site, e.g. HpaII sites are only 8% of the human genome. Other, more promiscuous restriction enzymes such as McrBC alleviate this to a degree, but still suffer from lack of specificity, as well as the difficulty and sample loss associate with purifying the cut fragments from the uncut. Alternatively, bisulphite pyrosequencing can determine the methylation status of the DNA, but this method is difficult to apply to large regions of DNA.
A number of conventional techniques are used for the purpose of enriching methylated DNA from unmethylated DNA. They include 1) immunoprecipitation of methylated DNA; 2) digestion using methyl-sensitive enzymes; 3) methylation sensitive PCR; and 4) DNA methylation binding columns. Those methods are able to enrich the DNA for either methylated or unmethylated fractions, but cannot actually sort methylated from unmethylated DNA piece by piece. All of those methods need large amounts of DNA to be effective, and have low yield in the sorting process. Of course, direct analysis of bisulfite converted DNA is also a possibility, through either sequencing (pyrosequencing or Sanger sequencing) or microarray analysis.
Immunoprecipitation of methylated DNA is based on the simple concept of using a monoclonal antibody raised against 5-methyl-cytosine (mC). Genomic DNA extracted from cells or tissue is fragmented through sonication or shearing to yield fragments on the order of 1 kbp or smaller. One fraction of the fragmented DNA is then denatured and immunoprecipitated with the mC antibody. The DNA is washed, and the precipitated DNA recovered, presumably highly enriched for fragments which have one or more methylated cytosines. Usually, this methyl enriched DNA is then run on a microarray versus the other fraction of the original “input” DNA from the sonication. The two DNA fractions are labeled with Cy3 and Cy5 fluorescent markers using standard kits, and the results assayed on the microarray to obtain a ratio (Weber et al. 2005 Nature Genetics 37: 853-62). This technique suffers from two complementary problems: unmethylated DNA is sometimes co-precipitated with methylated; and methylated DNA must be densely methylated in order to be extracted in the first place. In fact, it has recently been shown (Irizarry et al. 2008 “Comprehensive high-throughput arrays for relative methylation (CHARM)” Genome Research), that this technique is barely able to globally distinguish between a cell line with a double knockout of DNA methyltransferase 1 and 3B (DKO sample) and the HCT116 cell line, a hypermethylated colorectal cancer cell line.
Methyl-sensitive digestion is another method to sort methylated from unmethylated parts from the genome. Restriction enzymes are sequence specific, and some are sensitive to whether the DNA is methylated or not. One example is HpaII and MspI, a pair of isoschizomers recognizing CCGG, with MspI insensitive to methylated status and HpaII cutting only unmethylated recognition sites. Another unique enzyme currently being used more frequently is McrBC, which recognizes any pair of (A/G)mCs that are from 40-3000 bp apart, and cuts at one of the sites (Panne et al. 1999 Journal of Molecular Biology 290(1): 49-60). Genomic DNA is first fragmented, digested with the methylation sensitive enzyme, then either amplified with recognition site specific PCR or separated by size—either way purifying the fragments which are unmethylated, with the resulting fragments separated by size. Cleavage based ligation is also possible, using primers which match the recognition site and amplify only fragments which were cut—amplifying the unmethylated fraction, as in HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) (Khulan et al. 2006 Genome Research 16(8): 1046).
Methylation-specific PCR uses a combination of bisulfite treatment and careful primer design to determine methylation status of a given DNA locus. After bisulfite treatment, any unmethylated cytosine residue is deaminated, converting it to uracil. Methylated cytosines are protected from deamination, so they are kept as cytosines. This alters the sequence of the treated DNA in a predictable, methylation dependent way. By designing primers which amplify either unchanged cytosines or cytosines converted to uracil, the methylation status of the original genomic DNA is determined, based on which primers give product. This technique, while extremely specific and requiring relatively small amounts of genomic DNA (as few as 100 cells), does require extreme specificity—only one locus can be tested at a time for methylation, and the primer design requires specific knowledge of the area to be tested. Only one area can be tested at a time, requiring large amounts of DNA to test multiple sites, as well as long time periods and significant effort.
Separation can also be accomplished by taking advantage of the natural ability of certain DNA-binding proteins to differentiate between methylated and unmethylated DNA. By using a recombinant His-tagged version of the methyl binding domain (MBD) of MeCP2, and attaching it to a nickel-agarose matrix, a methyl sensitive stationary phase for a column is constructed (Cross et al. 1994 Nature Genetics 6(3): 236-244). Genomic DNA is then cleaved by MseI, which cuts in A/T heavy areas, yielding small CG rich fragments which may be run through the column and purified on the basis of the strength of binding. This allows for fractions to be extracted which vary in the level of methylation. The specificity of this technique is not ideal—as unmethylated DNA may also be bound by the column, and the strength of binding may vary depending on the degree of methylation. Dense methylation is needed for strong binding, essentially causing the same type of problem previously discussed with MeDIP.
Accordingly, provided herein are methods for separating DNA with high selectivity and specificity according to methylation profile and level in a relatively simple, fast and efficient manner without resorting to tags or enzymes. This is particularly advantageous in that such tags and enzymes are often expensive and difficult to implement without adversely impacting one or more of resolution, sensitivity, specificity, speed and selectivity.